A multi-adenylate cyclase regulator at the flagellar tip controls African trypanosome transmission

Signaling from ciliary microdomains controls developmental processes in metazoans. Trypanosome transmission requires development and migration in the tsetse vector alimentary tract. Flagellar cAMP signaling has been linked to parasite social motility (SoMo) in vitro, yet uncovering control of directed migration in fly organs is challenging. Here we show that the composition of an adenylate cyclase (AC) complex in the flagellar tip microdomain is essential for tsetse salivary gland (SG) colonization and SoMo. Cyclic AMP response protein 3 (CARP3) binds and regulates multiple AC isoforms. CARP3 tip localization depends on the cytoskeletal protein FLAM8. Re-localization of CARP3 away from the tip microdomain is sufficient to abolish SoMo and fly SG colonization. Since intrinsic development is normal in carp3 and flam8 knock-out parasites, AC complex-mediated tip signaling specifically controls parasite migration and thereby transmission. Participation of several developmentally regulated receptor-type AC isoforms may indicate the complexity of the in vivo signals perceived.

CARP3 is not essential for growth or differentiation of T. brucei but shows life cycle stage-specific localization (a) Cartoon representation of a predicted model of T. brucei CARP3 using AlphaFold 37 . Model confidence is illustrated using the predicted local-distance difference test (pLDDT) score, indicated by the color-coding. RNAi cell lines. RNAi was induced by addi i n L tetracycline (+Tet condition). Repression of CARP3 levels was confirmed by Western blot analysis (e) with PFR-A/C as loading control. Western blot corresponding to (d) is shown in Fig. 1b. (f, g) Growth of cell lines as in (d, e) during differentiation from BSF long slender (LS) to short stumpy (SS) (f) or during SS to PCF differentiation (g). Western blot in (f) shows e pression of CARP3 and the stumpy marker protein PAD1. PFR-A/C serves as loading control. The growth curve in (f) was started at time point 0 h with long slender cells at a density of 5-8 10 5 cells/mL and Tet induction of RNAi. Growth was monitored over 5 h without culture dilution, resulting in development into PAD1-e pressing short stumpy forms. The growth curve in (g) was initiated with the SS cells from (f). (h, i) ndirect immuno uorescence analysis of CARP3 (green) in T. brucei AnTat 1.1 procyclic forms (h) or long slender bloodstream forms (i). DNA was stained with DAP (blue). Fluorescence channels were merged with the differential in e e ence c n as . cale ba s .  (f) C C values distri utions for t o simulated independent Poisson point patterns characteri ed y densities e ual to the ones from procyclic T. brucei AnTat 1.1E expressing CARP3-PAmCherry and CARP3-m eon reen. The point patterns are con ned ithin a rectangular area of 1 m idth approximation of a straight flagellum .
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In vitro differentiation by RBP6 overexpression
Differentiation of procyclic forms by overexpression of RBP6 was performed in EATRO 1125 T7T as described previously 1,2 .

Cloning and generation of transgenic trypanosomes
Generation of a homozygous carp3 knock out: Both CARP3 alleles were deleted from AnTat 1.

Generation of a homozygous flam8 knock outs and rescue:
In AnTat 1.1E 'Paris' bloodstream forms, one flam8 allele was deleted and the second one truncated to generate flam8 knock-outs sub-clones as described in 6 . A rescue sequence was then re-introduced in the partially deleted allele to produce an add-back strain 6 .

Tetracycline-inducible RNAi of CARP3:
Two copies of a tetracycline repressor were integrated into the T.

C-terminal in situ tagging of CARP3 with YFP or mNeonGreen:
The CARP3 C-terminus was amplified from genomic DNA (strain AnTat 1.1) using primers p3074_CARP3_up_SwaI and p3074_CARP3_low_BamHI and cloned into the vector p3074 5 that enables C-terminal Ty1-tagging. The 4x Ty1-tag was swapped to YFP from p3329 using BamHI and EcoRI. For PALM microscopy, YFP from p3329 was replaced by mNeonGreen amplified from plasmid pK19msB-mNeonGreen-ptsG 8 by primers mNeon_BamHI_fw and mNeon_EcoRI_rev. The plasmids were linearized with NotI and transfected cells were grown in the presence of 2 µg/mL G418 or 0.1 µg/mL puromycin.

C-terminal in situ tagging of CARP3 with mCherry or photoactivatable (PA)mCherry:
For C-terminal fusion of CARP3 to mCherry, the long primer PCR tagging strategy was used 9 . YFP-Ty1 of plasmid pPOTv4 was replaced by mCherry-Ty1 followed by PCR amplification of mCherry-TY with primers pPOTv4_Lr_mCherry_TY_CARP3 fw and pPOTv4_Lr_mCherry_TY_CARP3 rev introducing stretches homologous to the C-terminus of the CARP3 ORF and the start of the CARP3 3'UTR. The PCR product was purified by phenol-chloroform extraction prior to transfection and selection was done with 2 µg/mL G418. For C-terminal fusion of CARP3 to photoactivatable (PA)mCherry, the same strategy was used. mCherry-Ty1 was replaced by PAmCherry amplified from plasmid pK19mobsacBparB-PAmCherry 10 using primers PAmCherry BamHI FWD and PAmCherry SacI REV.
Linearization was done with SphI and transfected cells were selected with 2 µg/mL hygromycin B.
A list of all primers used in this study is provided as Supplementary Table 2. All transfections were done in slender bloodstream forms, except for tagging of ACPs.

Social motility assay
Agarose plates for social motility assays were prepared as described 23 . 5 × 10 5 cells of T. brucei strain AnTat 1.1 or AnTat 1.1E were spotted in 5 µL of SDM-79 on agarose plates within 7 days after density-dependent differentiation from bloodstream to procyclic stage.

Generation of polyclonal antibodies
The CARP3 ORF was cloned as N-terminal His10 fusion into pETDuet-1 using primers Tb927.7.5340F10His and Tb927.7.5340_BamHI_rev via NcoI and BamHI restriction sites and transformed into E. coli Rosetta. 500 µg of His10-CARP3 purified using a Ni-NTA column (Qiagen) were used for immunization of rabbits by Eurogentec, followed by further boosts with 500 µg antigen. The CARP3 antiserum was affinity-purified using His10-CARP3 according to the method of Olmsted 24 .

Western blot
Western blot analysis was performed as previously described 19  .

Indirect immunofluorescence analysis
For microscopic analysis, trypanosomes were either spread on glass slides and fixed in methanol for 5 min at -20°C or fixed in 2% PFA for 20 min at room temperature.
Proteins were considered as statistically significant with FDR ≤ 0.05 and s0 = 2 (two- Cell lysis, protein digestion, peptide purification and MS/MS analysis were performed as described by Humphrey et al. 37 . Purified peptides were injected in an RSLCnano system (Thermo) and separated in a 25-cm analytical Aurora C18 nanocolumn (75 μm ID 120 Å, 1.6 μm, Ion Opticks) with a 120-min gradient from 4 to 40% acetonitrile in 0.1% formic acid. The effluent from the HPLC was directly electrosprayed into a Q Exactive HF (Thermo), operated in data dependent mode to automatically switch between full scan MS and MS/MS acquisition. Survey full scan MS spectra (from m/z 375-1600) were acquired with resolution R = 60000 at m/z 400 (AGC target of 3x10 6 ). The ten most intense peptide ions with charge states between 3 and 5 were sequentially isolated to a target value of 1x10 5  with the dataset identifier PXD025401.